A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six α-helices (α1-α6) out of which α2-α5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the α1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that α1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the α5 helix and the linker between α3 and α4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by α1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Shared components of mitochondrial and peroxisomal division

Mitochondria and peroxisomes are ubiquitous subcellular organelles, which are highly dynamic and display large plasticity. Recent studies have led to the surprising finding that both organelles share components of their division machinery, namely the dynamin-related protein DLP1/Drp1 and hFis1, which recruits DLP1/Drp1 to the organelle membranes. This review addresses the current state of knowledge concerning the dynamics and fission of peroxisomes, especially in relation to mitochondrial morphology and division in mammalian cells.     

Fis1, Mff,MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.     

Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis

During apoptosis, the mitochondrial network fragments. Using short hairpin RNAs for RNA interference, we manipulated the expression levels of the proteins hFis1, Drp1, and Opa1 that are involved in mitochondrial fission and fusion in mammalian cells, and we characterized their functions in mitochondrial morphology and apoptosis. Down-regulation of hFis1 powerfully inhibits cell death to an extent significantly greater than down-regulation of Drp1 and at a stage of apoptosis distinct from that induced by Drp1 inhibition. Cells depleted of Opa1 are extremely sensitive to exogenous apoptosis induction, and some die spontaneously by a process that requires hFis1 expression. Wild-type Opa1 may function normally as an antiapoptotic protein, keeping spontaneous apoptosis in check. However, if hFis1 is down-regulated, cells do not require Opa1 to prevent apoptosis, suggesting that Opa1 may be normally counteracting the proapoptotic action of hFis1. We also demonstrate in this study that mitochondrial fragmentation per se does not result in apoptosis. However, we provide further evidence that multiple components of the mitochondrial morphogenesis machinery can positively and negatively regulate apoptosis.     

Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.     

Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells

The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and mediates mitochondrial fission. Although the mitochondrial outer membrane (MOM) protein Fis1 is thought to be a Drp1 receptor, this has not been confirmed. To analyze the mechanism of Drp1 recruitment, we manipulated the expression of mitochondrial fission and fusion proteins and demonstrated that (a) mitochondrial fission factor (Mff) knockdown released the Drp1 foci from the MOM accompanied by network extension, whereas Mff overexpression stimulated mitochondrial recruitment of Drp1 accompanied by mitochondrial fission; (b) Mff-dependent mitochondrial fission proceeded independent of Fis1; (c) a Mff mutant with the plasma membrane-targeted CAAX motif directed Drp1 to the target membrane; (d) Mff and Drp1 physically interacted in vitro and in vivo; (e) exogenous stimuli-induced mitochondrial fission and apoptosis were compromised by knockdown of Drp1 and Mff but not Fis1; and (f) conditional knockout of Fis1 in colon carcinoma cells revealed that it is dispensable for mitochondrial fission. Thus, Mff functions as an essential factor in mitochondrial recruitment of Drp1.     

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.     

Fine-tuning of Drp1/Fis1 availability by AKAP121/Siah2 regulates mitochondrial adaptation to hypoxia

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C.?elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.     

Targeting of hFis1 to peroxisomes is mediated by Pex19p

The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.     

Control of mitochondrial morphology through differential interactions of mitochondrial fusion and fission proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria.     

Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulate cellular senescence

The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated beta-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated beta-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.     

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.     

Regulation of Ca2+-induced permeability transition by Bcl-2 is antagonized by Drp1 and hFis1

The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca²⁺. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca²⁺ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1. The complexity of protein–protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.     

The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis

Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.     

Mutations in Fis1 disrupt orderly disposal of defective mitochondria

Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1^−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes.     

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six α helices [α1–α6] out of which [α2–α5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the α1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[α2–α6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the α2 and α3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the α1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of α1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.